First published online March 31, 2007
Journal of Experimental Biology 210, 1455-1462 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.02756
Neuroprotection from secondary injury by polyethylene glycol requires its internalization
Peishan Liu-Snyder1,
Melissa Peasley Logan1,
Riyi Shi1,2,
Daniel T. Smith3 and
Richard Ben Borgens1,2,*
1 Center for Paralysis Research, School of Veterinary Medicine, Purdue
University, West Lafayette, IN 47907, USA
2 Weldon School of Biomedical Engineering, College of Engineering, Purdue
University, West Lafayette, IN 47907, USA
3 Department of Industrial and Physical Pharmacy, Purdue University, West
Lafayette, IN 47907, USA
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Fig. 1. Cell death after polyethylene glycol (PEG) application to acrolein-treated
PC12 cells. (A–C) Control PC12 cells are shown developing on the culture
substrate. Notice that these cells were well attached and some have extended
processes giving them a stellate appearance. (A') The same culture as in
A, 4 h after a `sham treatment' – the introduction of PBS into the
culture medium. Notice that all cells in this field of view are healthy,
developing and all together similar to the culture 4 h previously. (B')
The same culture as in B, 4 h after the introduction of 100 µmol
l–1 acrolein. Note the significant loss of cells with cell
debris littering the substrate. (C') The same culture as in C, showing
significant cell death as a result of acrolein treatment, in spite of the
addition of 10 mmol l–1 PEG to the culture medium within 15
min after acrolein.
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Fig. 2. Acrolein, mitochondrial functioning and polyethylene glycol (PEG).
Absorbance (ordinate) is an index of mitochondrial function/oxidative
metabolism. Note that control cells (untreated) showed characteristic MTT
(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) absorbance
spectra and the addition of PEG to the medium did not affect the MTT assay.
Mitochondrial function, however, is markedly compromised by the addition of
100 µmol l–1 acrolein, and this result is not affected by
the inclusion of PEG in the assay medium. *Statistically significantly
different from the control (P 0.05).
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Fig. 3. Polyethylene glycol (PEG) accumulates on the outside of PC12 cells. (A) 1 h
after the addition of fluorescently decorated PEG, 3D confocal microscopy
revealed that the only labeling above the limit of detection was confined to
the surface of acrolein-treated cells. (B) By 2 h post treatment this same
result was obtained, whereas at 3 h incubation time (data not shown), PEG was
beginning to become apparent within the cells. These 2D representations,
captured from 3D animated data sets, do not easily reveal the location of
fluorescence when confined to the surface. Animating the 3D reconstruction,
however, confirmed this surface labeling.
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Fig. 4. The effects of polyethylene glycol (PEG) on CAP recovery following
ischemia/acrolein reperfusion. (A) The double-sucrose gap-recording chamber.
The device is constructed of PlexiglasTM with five separate chambers, all
linked together by a narrow slot. Three large chambers are shown; the two end
ones filled with KCl and the middle chamber with a physiological medium (a
modified Krebs' solution), where a constant flow of medium through the chamber
is carried out. The delivery to an `antechamber' reduces any turbulence or
artifacts based on flow of medium through and out of the device. A delivery
and an aspiration tube (that sets the fluid level) are shown for the central
chamber and one of the small sucrose chambers to its right. A constant flow of
sucrose significantly reduces the mixing of the KCl solution in the end
chambers with the center one. A full length ( 40 mm) of guinea pig spinal
cord or a wedge-shaped long strip of only ventral white matter was placed
across and within all five chambers. The ends of the dissected cord are then
near intracellular potential, while the center of the cord is near
extracellular potential. Bipolar stimulating electrodes fire compound action
potentials (CAPs) at one end of the cord, and these are recorded arriving at
the other end with bipolar recording electrodes. This arrangement provides a
very precise recording of CAPs in cord for many hours at a time. It also
allows the addition of test drugs or other interventions to be carried out in
the central chamber. (B) The CAP amplitude profile in (a) the presence of
oxygen and glucose deprivation (OGD), (b) OGD plus acrolein, and (c) OGD plus
acrolein plus PEG. This graph displays the CAP amplitude recorded over a
period of time. The values were normalized. Note the similarity in CAP
amplitude recovery during the reperfusion period for groups (b) and (c). (C)
The CAP waveforms are shown at three time points, (a) pre OGD, (b) at the end
of OGD and (c) at the end of reperfusion, as indicated in B. Note there is
little difference from the initial CAPs and the ones recorded following
recovery from ischemia in OGD group. However, the amplitude is reduced to a
half of pre-OGD levels in both groups (b) and (c). Scale bars, 1 mV, 1 ms.
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Fig. 5. Compound action potential (CAP) recovery in oxygen and glucose deprivation
(OGD), OGD plus acrolein, and OGD plus acrolein plus polyethylene glycol (PEG)
groups. In the OGD alone group, there was a 97.6±7% recovery of the CAP
following an ischemic insult (N=8). The CAP recoveries of the OGD
plus acrolein and the OGD plus acrolein plus PEG groups were 46±5% and
45.7±4.6%, respectively. N=6 in each group. *P=0.88;
Student's t-test, unpaired, two tail.
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© The Company of Biologists Ltd 2007
